Haematology: Haematopoiesis, Erythropoiesis, Anemia

Cellular and Transfusion Science - Haematology Lecture 1

• The lecture covers Haematopoiesis, Erythropoiesis, Anaemia Classifications and Anaemias

Haematopoiesis

• This includes the principles of blood cell type development

Stem Cell Hypothesis

• This includes the principles of self-renewal and commitment

Erythropoiesis

• This includes the development of red blood cells

Blood Cell Concentrations

• Red blood cells (erythrocytes) have a typical concentration of 5 × 10^12 cells/liter in human blood and transport O2 and CO2
• Neutrophils (polymorphonuclear leucocytes) have a concentration of 5 × 10^9 cells/liter and phagocytose/destroy bacteria
• Eosinophils have a concentration of 2 × 10^8 cells/liter and destroy larger parasites, modulating allergic inflammatory responses
• Basophils have a concentration of 4 × 10^7 cells/liter and release histamine (and serotonin in some species) in certain immune reactions
• Monocytes have a concentration of 4 × 10^8 cells/liter and become tissue macrophages, digesting microorganisms and foreign bodies
• Lymphocytes: B cells have a concentration of 2 × 10^9 cells/liter and make antibodies
• Lymphocytes: T cells have a concentration of 1 × 10^9 cells/liter and kill virus-infected cells and regulate activities of other leucocytes
• Natural killer (NK) cells have a concentration of 1 × 10^8 cells/liter, killing virus-infected and some tumor cells
• Platelets (cell fragments from megakaryocytes in bone marrow) have a concentration of 3 × 10^11 cells/liter and initiate clotting
• Humans have about 5 liters of blood, accounting for 7% of body weight
• Red blood cells constitute about 45% of blood volume, with white blood cells about 1%; the remainder is plasma

Stem Cell Theory

• A stem cell is unspecialized, capable of self-renewal and developing into specialized cells
• Progenitor cells are unspecialized or partially specialized, undergoing division to yield two specialized cells

Cell Differentiation Definitions

• TOTIPOTENT: form all cells including extraembryonic and placental cells
• PLURIPOTENT: give rise to all cell types
• MULTIPOTENT: give rise to more than one cell type but limited
• All blood cells have a common ancestral cell - MULTIPOTENT STEM CELL (MSC)
• MSC can differentiate to Colony Forming Unit-Granulocyte, Erythrocyte, Monocyte, Megaokaryocyte (CFU-GEMM), myeloid cell line (late RBC's, platelets, granulocytes and monocytes), or lymphoid stem cell (lymphoid cell line – lymphocytes and natural killer cells)
• All cells derive from a self-renewing pool of stem cells
• Pluripotential and multipotential stem cells give rise to committed stem cells for each cell line
• Committed stem cells have receptors for specific growth factors
• Cells respond to stimulation by division and maturation (precursor cell stages) into end-stage cells

Hematopoietic Stem Cells (HSC)

• Stem cells are capable of self-renewal and multi-lineage differentiation
• HSC are multi-potent and occur at a frequency of 1:5000 in bone marrow
• HSCs can be defined by their capacity to give rise to non-self-renewing populations that generate multiple terminally-differentiated cell types, and by their surface phenotype: c-kit+Sca-1+Lin-Thy1lo

Cell Fate Decisions

• Cell fate is determined by interactions between extrinsic (soluble growth factors) and intrinsic factors (transcription factors).
• Transcription factors such as Runx1 (required for normal hematopoiesis), Ikaros (regulator of immune system development), GATA-1 (essential for erythroid and platelet forming cells), Pax5, E2A, EBF, and Notch-1/GATA-3 (T cell development) are key in specifying cell fate decisions.

Development of Blood Cells

• At 3 weeks, the formation of blood islands begins in the yolk sac.
• By 6 weeks, the liver becomes the primary hematopoietic organ.
• From 6-8 weeks, the spleen functions in hematopoiesis until the 8th month.
• Around 20 weeks, bone marrow takes over and remains the primary site throughout life.

Stem Cell Niches

• These exist within the Bone Marrow
• Stem cell niche is a specific site in adult tissues where stem cells reside
• Stem cells undergo renewal and differentiation in the niche
• Bone marrow is home to two niches, the Osteoblastic niche (at the endosteal surface) and the Vascular niche (involving sinusoidal blood vessels)
• These niches balance self-renewal and differentiation
• Osteoblastic niche maintains quiescence and harbors the Long Term-HSC
• Vascular niche supports proliferation, differentiation and mobilization (transendothelial migration) of Short Term-HSC to the blood stream in response to physiological demands and back up during BM stress

Erythropoiesis: Generation of Red Blood Cells

• The erythroid maturation sequence consists of several stages from early to late development
• The stages are: Proerythroblast (Pronormoblast) → Basophilic Normoblast → Polychromatophilic Normoblast → Orthochromatophilic Normoblast → Reticulocyte → Erythrocyte
• The first cell committed to becoming an RBC is the Proerythroblast
• The nucleus becomes smaller to form the basophilic erythroblast. Cytoplasm becomes more basophilic due to ribosomes
• More hemoglobin is produced to form the polychromatophilic erythroblast. Cytoplasm displays both basophilic and eosinophilic stains
• The nucleus extrusion forms the orthochromatophilic erythroblast, and the reticulocyte enters the circulation
• The reticulocyte has reticular networks of polyribosomes in its cytoplasm

Reticulocytes

• These are defined as Young red blood cells
• They contain small amounts of RNA
• They undergo removal of RNA on passing through the spleen in 1st day of life
• Generally stain more blue than mature RBC's on Wright stain (polychromatophilic)
• Generally slightly larger than mature RBC
• They are an important marker of RBC production
• Reticulocyte count = Retic % x RBC Count, e.g., 0.01 x 5,000,000 = 50,000 (Normal: up to 100,000)

Erythropoiesis Location

• It is in the bone marrow
• HSCs differentiate into BFU-E (burst forming unit-erythroid), then CFU-E (colon forming unit-erythroid), ProE (proerythroblast), BasoE (basophilic erythroblast), PolyE (polychromatophilic erythroblast), and OrthoE (orthochromatophilic erythroblast)
• Hemoglobin accumulates, nuclear condensation occurs, cell size and mRNA decrease, and morphology becomes identifiable
• Enucleation occurs before blood stream entry as a reticulocyte

Erythropoiesis Factors

• This includes intrinsic and extrinsic factors
• Transcription factors, small RNAs, and DNA-binding factors influence the process
• Stem Cells differentiate into CFU-GEMM, then BFU-Es, and CFU-Es before becoming erythroblasts
• Growth Factors: IL3, SCF, GM-CSF, BMP4, +/-Epo

Erythropoietin

• Hormone produced in peritubular fibroblast-like cells consists of 165 amino acids glycosylated with 4 carbohydrate chains
• Erythropoietin prevents apoptosis
• Erythropoietin levels increase with decreasing hemoglobin levels
• Erythropoietin works the following way in the body: Lack of oxygen --> kidney --> Erythropoietin --> Bone Marrow --> Erythrocytes

Red Blood Cells

• Normal red blood cells are anucleate, highly flexible biconcave discs with an 80-100 femtoliters volume
• Red blood cells are highly flexible for passage through capillaries
• They carry oxygen to and carbon dioxide away from cells
• Red blood cells circulate for approximately 120 days.
• Macrophages can break down dead red blood cells in the spleen, liver, or red bone marrow
• Heme is split into biliverdin, then bilirubin
• Amino acids from globin are reused for protein synthesis
• Iron is stored in liver
• In the bone marrow, the process of erythropoiesis produces new red blood cells

Red Blood Cell Metabolism

• Metabolize without a nucleus and mitochondria
• The Embden-Meyerhof Pathway, Pentose phosphate pathway and Methemoglobin reductase pathway are key metabolic pathways for cells
• The Embden-Meyerhof Pathway involves glycolysis and lactic acid fermentation
• The Pentose phosphate generates reduced NAD i.e. NADPH. NADPH generates reduced glutathione (anti-oxidant)
• The Methemoglobin reductase pathway maintains iron in Fe2+ state

Anaemia

• This can be defined as Haemaglobin < 13g/dL (men), <12g/dL (women), <11g/dL if pregnant
• The Aim is to describe the classification and characteristics of classifications
• Reference range 13.3-16.7g/dL men, 11.8-14.8g/dL women

Etiology

• Causes vary
• They include: decrease in production, increase in destruction and blood loss
• Classification exists for cell size and in clinical signs and genetics
• This can be diagnoses with full blood count and reticulocyte count
• Can be relative (dilutional effects e.g. increased plasma volume/pregnancy) or absolute
• Blood disorders arise due to disturbances in DNA synthesis, enzyme defects
• Some include mechanical chemical and infection, with some being intrinsic or extrinsic
• This can be caused by issues related with the bone marrow, with globin synthesis being one cause
• Iron parameters and the thyroid are critical to determine.

Classification of Anaemias

• This can be determined by film appearance and underlying pathology
• Appearances include: Normochromic/normocytic (NN), Microcytic (hypochromic) (MH) and Macrocytic (normochromic) (MN)
• This can also be measured under underlying pathology such as increase destruction, increase loss and decrease production
• Can be diagnosed with physical examination, full blood count, Reticulocyte count and bone smear biopsy.

Diagnosis symptoms

• Weakness, Shortness of breath and Pallor
• Fatigue, Palpitations and Tachycardia (pulse >100/min)
• Nervous system involvement, parasthesia and Jaundice
• Koilonychia and dark urine alongside to Dizziness
• Age, Symptoms, History of glossitis and Nervous involvement
• There will dietary intake (?? alcohol), residence in tropics, drug therapy, abdominal surgery and pregnancy symptoms
• Assessment of red blood cell includes, General nutritional state, tongue conditions, abdomen conditions and operation scars

RBC Assessment

• Determined on number, size, shape and colour
• RBC Size - Large, normal size, or small as well as variable sizes (anisocytosis)
• Normal biconcave discs. versus spherocytes, versus oddly shaped cells
• RBC Color - Generally an artifact of size of cell

Heamatology Blood Count

• Haemaglobin levels determine if anaemia
• Red blood count are important
• Key measurements are: Haematocrit; %age of RBC, MCV-average volume of red cells-size, MCH- average content weight of Hb/RBC and MCHC-average weight of hemoglobin per unit volume of the red blood cell

Normocytic Anaemia

• This displays a normal RBC size and a reduced number
• Involves a reduction in Haemaglobin (Hb) and RBC counts
• A normal mean corpuscular volume (MCV) of 80-100 Femto litres, mean corpuscular haemaglobin and concentration will also be measured.
• This Is due too bleeding to bone destruction

Macrocytic Anaemia

• This is the same as (normochromic) (MN) anaemias
• MCV of > 100 fl and a normal MCHC
• This involves cells that are much larger
• Involves Megaloblastic and haemolytic anaemias

Microcytic (hypochromic) Anaemia

• (MH) anaemias will have a MCV of < 80 fl and a MCHC of < 30 g/dl
• Main causes will be iron deficiency anaemia and Thalassaemia

Anaemia due to haemorrhage.

• Shows blood loss due to the production of fluid to restore loss
• The start to RBC regeneration will show increase in reticulocytes
• Increased WBC
• As iron store become more depleted.

Aplastic Anaemia

• This is not very functional
• Will show as Congenital and infections and Autoimmune issues
• It can be treated with multiple transfusions

Haemolytic Anaemia

• Results from an increase in the rate of red blood cell destruction
• Results can have a varying degrees due to some external event.
• The spleen is critical to remove the Haemolytic events
• Key Indicators will be found in spleen and liver.